Proglumide and pharmaceutical compositions containing it for use in the treatment of neoplastic affections

ABSTRACT

This inventions relates to a method of treating malignant tumors of the pancreatic, colic or gastric origin whose growth is increase by bioactive polypeptides comprising administering an anti-tumor effective amount of proglumide or pharmaceutical acceptables salt thereof to a patient afflicted with said malignant tumors.

The subject of the present invention is a new therapeutic use of D,L-4-benzamido-N,N-dipropyl-glutamic acid (proglumide) and its pharmaceutically-acceptable salts, for use in the treatment of neoplastic affections in which there is a pathological cell increase indirectly affected by gastrin, cholecystokinin (CCK), other bioactive peptides or by related mechanisms which are not yet well clarified.

The applicants have found that the said drug, already widely used in the treatment of gastric ulcers (see for example Merck Index No. 7680, 10th edition), in fact has an unexpected but extremely interesting therapeutic activity, which is that of significantly inhibiting the cell increase of tumour cells resulting from chemically induced pancreatic, colic or gastric cancer or of destroying such cells.

The invention is based on the discovery that the polypeptide hormone gastrin has a trophic effect on the digestive epithelium, just as the hormone CCK has a trophic action on pancreatic acini. Thus the chronic administration of gastrin to rats (or of pentagastrin which is the biologically active part of the physiological hormone) causes hyperplasia of the fundic and colic mucous membranes. Recently (Winsett et al--Surgical Forum, 33, 384 (1982)) it has been shown that gastrin stimulates the growth, in mice, of transplantable colic tumours obtained by chemical induction.

It has also been shown that the chronic treatment of rats with cerulein (a synthetic analog of CCK) causes hyperplasia of the pancreatic acinus cells (Solomon et. al.--An. J. Physiol 235, E714-719 (1978)).

On the basis of these theoretical premises, there has been a desire to experiment to see whether proglumide might behave as an antagonist to the growth of induced gastro-intestinal tumours and to evaluate whether this action could also affect the survival time of the animals.

This antitumoral activity of proglumide, which is the subject of the present invention, will thus be illustrated by a series of pharmacological experiments in vitro and in vivo arranged to show both the qualitative and quantitative aspects of the antitumoral activity and the mechanism by which this activity is manifested.

Experiment 1 Action of Proglumide on the rate of pentagastrin-induced growth of normal and tumoral colic cells

Male mice having a weight of 20-25 g were innoculated subcutaneously in the interscapular region with a suspension of 8×10⁴ colic-adenocarcinoma tumour cells taken from a mouse.

Four groups of 12 animals were used, that is: a group of control animals, a group of animals treated with 200 mg/kg i.p. doses of proglumide 3 times a day, a group of animals treated with 200 μg/kg of pentagastrin every eight hours and a group of animals treated with proglumide and pentagastrin in the manner indicated above.

After 20 days the animals were killed and the fundic mucous membranes and tumours were removed, weighed and extracted to determine the DNA. The results obtained are given in Table 1.

                                      TABLE 1                                      __________________________________________________________________________     Antagonistic activity of Proglumide towards the growth of colic                tumours induced by pentagastrin                                                                  Weight of             weight of                                      No        the mucous            the colic                                      Ani-      membrane                                                                             student's                                                                           fundic                                                                               student's                                                                           tumour                                                                               student's                                                                           tumour                                                                               student's             GROUPS  mals                                                                              Dose   (mg)  t    DNA (mg)                                                                             t    (mg)  t    DNA (mg)                                                                             t                     __________________________________________________________________________     A: Control                                                                             12   --   15.9 ± 1.44                                                                       --   0.21 ± 0.02                                                                       --   500 + 12.6                                                                             -- 2.90                                                                                 --0.23                B: Pentagastrin                                                                        10.sup.(°)                                                                 200 mcg/kg-3                                                                          40.9 ± 1.26                                                                       12.78.sup.(***)                                                                     0.49 ± 0.03                                                                       9.46.sup.(***)                                                                      800 ± 12.8                                                                        16.6.sup.(***)                                                                      4.56                                                                                 3.37.sup.(**)         (PEG)      per day                                                             C: Proglumide                                                                          12 200 mcg/kg-3                                                                          13.3 ± 1.58                                                                        1.21                                                                               0.12 ± 0.02                                                                       3.17.sup.(**)                                                                       470 ± 11.5                                                                         1.75                                                                               2.54                                                                                 1.10 0.23             (PR)       per day                                                             D: PEG + PR                                                                            12 200 mcg/KG                                                                            16.0 ± 1.27                                                                       vsA: 0.21 ± 0.05                                                                       vsA:0                                                                               519 ± 11.8                                                                        vsA: 2.80                                                                                 vsA: 0.24                        PEG +         0.05      vsB:        1.11      0.32                             200 mg/kg    vsB:       4.43.sup.(***)                                                                            vsB:       vsB:                             PR           13.7.sup.(***)        16.1.sup.(***)                                                                            3.57.sup.(**)         __________________________________________________________________________      .sup.(°) 2 animals died during the treatment                            .sup.(**) P < 0.01                                                             .sup.(***) P < 0.001                                                     

The data given in the table show that PEG causes both significant hyperplasia of the fundic mucous membrane and a significant increase in the weight of the mucous membrane tumour and of the DNA content. However proglumide, at the doses used, is capable of inhibiting significantly both of the trophic actions of pentagastrin.

Experiment 2 Antagonism of proglumide to the rate of incorporation of tritiated thymidine in colic tumour cells, stimulated and unstimulated by pentagastrin

The groups of animals and the treatments were identical to those given in the preceding experiment. On the seventh day, two hours after the final treatment, the animals were killed and the tumours which had developed were explanted.

Small pieces of tissue were incubated at 37° for 30 minutes in a Dulbecco-modified Eagle-type culture medium together with 2 μCi of (3-H)-thymidine. The reaction was interrupted by the addition of 0.4N perchloric acid containing carrier thymidine. The samples were homogenised with 2 ml of 0.2N perchloric acid and RNA and proteins were removed by conventional techniques.

The DNA content of the samples was determined by the colorimetric method with diphenylamine (Biochem. J. 62, 315-323 (1956)).

The incorporation of the (3-H) thymidine in the DNA was determined by counting coupled aliquots of filtrate in a liquid scintillator under conditions suitable for counting the tritium.

The results were expressed as disintegrations per minute per microgram of DNA.

The results obtained are given in Table No. 2 and are expressed as a percentage variation with respect to the controls of the dpm of tritiated thymidine per mcg of DNA.

                                      TABLE 2                                      __________________________________________________________________________     Influence of Proglumide and Pentagastrine on the                               incorporation of tritiated thymidine in the synthesis of                       DNA in cultures of tumour cells in vitro.                                                        % VARIATION                                                                    WITH RESPECT                                                                   TO THE CONTROL                                                                             STUDENT'S                                                          GROUP       t                                                GROUPS    DOSE    (mean ± S.E.)                                                                           (P)                                              __________________________________________________________________________     A: Controls       100.0 ± 4.9                                               B: Pentagastrin                                                                           200 mcg/kg                                                                             285.0 ± 20.3                                                                           vsA:8.859                                        (PEG)     (3 per day)         (<0.001)                                         C: Proglumide (PR)                                                                        200 mg/kg                                                                              88.2 ± 5.2                                                                             vsA:1.65                                                   (3 per day)                                                          D: (PEG) + (PR)                                                                           200 mcg PEG                                                                           112.0 ± 8.4                                                                             vsA:1.24                                                   (3 per day)         vsB:7.88                                                   +200 mg PR          (<0.001)                                                   (3 per day)                                                          __________________________________________________________________________

From the data given in the table it is seen that pentagastrin accelerates the replication of DNA in colic tumour cells; in fact, at the doses used, the incorporation of tritiated thymidine, is found to be increased by about three times compared to the controls, an increase which, on a statistical basis, is highly significant.

Proglumide alone reduces the incorporation but not significantly, while the combined treatment with pentagastrin and proglumide means that the antagonistic action of the latter prevents the pentagastrin from reaching the target tissue and there is thus a clear reduction (statistically significant) in the incorporation of tritiated thymidine in the DNA compared with the group of animals treated solely with pentagastrin.

Experiment 3

In order to check the possibility that proglumide can influence the survival time of animals having a transplanted colic adenocarcinoma, the following experiment was carried out.

Male mice having a weight of 20-25 g were innoculated subcutaneously, in the interscapular region, with a suspension of 1×10⁵ colic-adenocarcinoma tumour cells taken from a mouse. Four groups of 12 animals were used, that is: a group of control animals and three groups of animals treated with 100, 200 and 400 mg/kg i.p. doses of proglumide respectively, three times a day.

This treatment was continued up to the 35th day, the day on which the final control animal died. The straight line of regression for the survival time of the various groups, starting from the first day on which a death occurred was calculated by the least squares method. It was thus possible to calculate an ED50, that is, the single daily dose of the drug capable of increasing the survival time of the animals by 50%.

The results obtained are given in Table No. 3.

                                      TABLE 3                                      __________________________________________________________________________     Effect of Proglumide on the survival time of mice innoculated                  with colic adenocarcinoma.                                                                                          CALCULATED    DAYS OF                                                          STRAIGHT LINE OF                                                                             LIFE PER                            DAYS ON WHICH DEATHS (INDICATED IN                                                                     (%)  REGRSSION (1) AND                                                                            ANIMAL                              PARENTHESES OCCURRED)   DEATH                                                                               COEFFICIENT OF                                                                               (AVER-                      TREATMENT                                                                              starting from time 0.   TOTAL                                                                               CORRELATION (r)                                                                              AGE)  ED60                  __________________________________________________________________________     Control group                                                                          17(1)-20(1)-22(2)-24(1)-26(2)-29(1)-30(1)-32(2)-35(1)                                                  100  n° animals = 22.35                                                                    25.3647                                                                              --                                                         (r = 0.995)                               Proglumide                                                                             20(1)-24(1)-27(2)-29(1)-30(1)-33(1)-34(2)                                                              75   n° animals = 22.47                                                                    30.1545                                                                              342.9                 100 mg/kp i.p.                       (r = 0.98)          (r = 0.99)            Proglumide                                                                             22(1)-23(1)-25(1)-28(1)-30(2)-33(1)-35(1)                                                              66.6 n° animals = 23.06 - 0.55                                                             31.5                        200 mg/kg i.p.                       (r = 0.99)                                Proglumide                                                                             23(1)-28(1)-31(1)-33(1) 33.3 n° animals = 18.82 - 32                                                               40.4                        400 mg/kg i.p.                       (r = 0.98)                                __________________________________________________________________________      (1) The straight lines of regression were calculated from the day at whic      the first death occurred.                                                

From an examination of the table it is seen that the treatment with proglumide significantly increases, and in a dose dependent manner, the survival time of animals innoculated with colic adenocarcinoma. This effect is probably related to the antagonistic action of proglumide towards endogenous gastrin which, as previously explained, has a stimulating effect on the growth of gastrointestinal tumour cells. An ED50 value of about 340 mg/kg was also calculated, this being the dose which enables the survival time of the animals to be doubled.

Experiment 4 Inhibiting action of proglumide on the rate of growth of pancreatic adenocarcinoma induced by CCK-8

Male hamsters were innoculated in the cheek pouch with a suspension of 1×10⁵ pancreatic adenocarcinoma tumour cells. After five days from the innoculation the animals were divided at random into four groups of 10 animals each, that is a control group, a group of animals treated with 250 mg/kg i.p. of proglumide three times a day, and a fourth group treated with proglumide and CCK-8 in the manner described above.

After 15 days of this treatment the animals were killed and the normal pancreas and the pancreatic tumours introduced into the cheek pouches were removed and weighed. The DNA was extracted and measured by conventional techniques.

The results obtained are given in Table 4 where they are expressed as average values±S.E.

The data given in the Table show that the hormone cholecystokinin (of which CCK-8 is the biologically active component), which has a trophic action on normal pancreatic cells, also stimulates the growth of pancreatic adenocarcinoma. Proglumide, which is a specific antagonist to CCK, antagonises both these actions of CCK-8 to a highly significant extent.

The experimental data given above seem fully to support the theory that the use of proglumide may be particularly favourable in the treatment of neoplasia in general and, more particularly, of that which is certainly sustained by endogenous bioactive polypeptides (such as gastrin and CCK), such as gastrointestinal and pancreatic neoplasia.

This treatment may in fact be extremely advantageous in that the rate of proliferation of these tumour cells has been shown to be stimulated and sustained by polypeptides, such as gastrin and cholecystokinin, for which proglumide is a specific antagonist in target cells, that is in gastric cells, colic cells and in the pancreatic acinus. Thus by antagonising these hormones in a selective manner, proglumide may act advantageously in blocking the indiscriminate development of these neoplastic forms or even causing their regression. This antagonism has been shown by various techniques, both in vivo and in vitro, by the study of the incorporation of tritiated thymidine in the DNA, which incorporation occurs in the S phase of the cell cycle, a phase on which the polypeptide hormones seem to exercise their trophic action.

It has also been shown that proglumide has an effective action even on the endogenous component of these hormones as shown by the increase in the survival time of animals in which colic adenocarcinoma had been implanted and which were treated with proglumide. Pharmaceutical forms including proglumide for use in the treatment of neoplastic affections are of conventional type. They may include pharmaceutically-acceptable inactive ingredients, such as binders, excipients, dispersants, preservatives, humectants and dyes possibly in association with drugs having an antimitotic action.

                                      TABLE 4                                      __________________________________________________________________________     Inhibiting action of Proglumide on the rate of growth of normal                and tumoral pancreatic cells induced by CCK-8.                                                                        WEIGHT                                                                         OF THE                                                   WEIGHT                PANCRE-                                                  OF THE     PANCRE-    ATIC                                            N°                                                                               PAN-       ATIC       CARCI-                                          Ani-     CREAS student's                                                                           DNA   student's                                                                           NOMA   student's                                                                           TUMORAL                                                                               student's            TREATMENT                                                                              mals                                                                              DOSE  (mg)  t    (mg)  t    (mg)   t    DNA    t                    __________________________________________________________________________     A: Control                                                                             10   --  380 ± 29.7                                                                          -- 0.8 ± 0.10                                                                          -- 113 ± 7.66                                                                           -- 0.4 ± 0.05                                                                           --                 B: CCK-8                                                                                8.sup.(°)                                                                  10 mcg/kg                                                                           600 ± 54.1                                                                        3.76.sup.(**)                                                                       1.5 ± 0.11                                                                        4.72.sup.(***)                                                                      180 ± 15.2                                                                         4.17.sup.(***)                                                                      0.7 ± 0.13                                                                         2.79.sup.(*)                    (3 per                                                                         day)                                                                C: Proglumide                                                                          10 250 mg/kg                                                                            330 ± 40.7                                                                        0.99 0.9 ± 0.13                                                                        0.61 100 ± 8.6                                                                          1.15 0.4 ± 0.06                                                                         0                    (PR)       (3 per                                                                         day)                                                                D: PR + CCK-8                                                                          10  10 mcg/kg                                                                           370 ± 38.0                                                                        vsA:0.2                                                                             1.0 ± 0.09                                                                        vsA:1.56                                                                            108 ± 8.8                                                                          vsA:0.45                                                                            0.4 ± 0.09                                                                         vsA:0                           CCK-8 +     vsB:       vsB:        vsB:        vsB:                            250 mg/kg   3.58.sup.(**)                                                                             3.56.sup.(**)                                                                              4.3.sup.(***)                                                                              2.33.sup.(*)                    PR                                                                  __________________________________________________________________________      .sup.(°) 2 animals died during the treatment                            .sup.(*) P < 0.05                                                              .sup.(**) P < 0.01                                                             .sup.(***) P < 0.001                                                      

We claim:
 1. A method of treatment of malignant tumors whose growth is increased by bioactive polypeptides comprising administering an anti-tumor effective amount of proglumide or a pharmaceutically-acceptable salt thereof to a human patient afflicted with said malignant tumors, wherein said malignant tumors are of pancreatic, colic or gastric origin.
 2. The method as claimed in claim 1, wherein said bioactive polypeptides are selected from the group consisting of gastrin and cholecytokinin. 